A CRISPR-Cas12a-Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application
Related Experiment Video
Updated: Aug 11, 2025
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Nan Jia1, Juan Zhou1, Fei Xiao1
1Experimental Research Center, Capital Institute of Pediatrics, Beijing, China.
Abstract:
Mycoplasma pneumoniae (MP), which is responsible for a majority of community-acquired pneumonia (CAP) in children, has been largely underestimated. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform termed MP-MCDA-CRISPR assay for MP infection diagnosis and clinical application. The MP-MCDA-CRISPR assay amplified the CARDS gene of MP by MCDA method, followed by trans-cleavage of the reporter molecular upon the formation of CRISPR-Cas12a-gRNA-target DNA complex, which was confirmed by the release of fluorescent signals. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the CARDS gene of MP. The optimal temperature for MCDA pre-amplification is 64°C, and the time for CRISPR-Cas12a-gRNA biosensing process is 5 min. The limit of detection (LoD) of the MP-MCDA-CRISPR assay is 50 fg per reaction without any cross-reaction with other non-MP pathogens. The MP-MCDA-CRISPR assay accurately identified the 50 real time-PCR positive clinical samples and 78 negative ones. Taken together, the MP-MCDA-CRISPR assay designed here is a promising diagnostic tool for point-of care (POC) testing of MP infection.
Insights
A new MP-MCDA-CRISPR assay offers rapid and accurate detection of Mycoplasma pneumoniae (MP) infection. This innovative diagnostic tool shows promise for point-of-care testing in children with pneumonia.
Area of Science:
- Molecular Biology
- Biotechnology
- Infectious Diseases
Background:
- Mycoplasma pneumoniae (MP) is a primary cause of community-acquired pneumonia (CAP) in children, often underestimated.
- Accurate and rapid diagnostics are crucial for effective MP infection management.
Purpose of the Study:
- To develop a novel, sensitive, and specific diagnostic assay for Mycoplasma pneumoniae (MP) infection.
- To evaluate the clinical applicability of the developed assay for point-of-care (POC) testing.
Main Methods:
- Coupling multiple cross displacement amplification (MCDA) with a CRISPR-Cas12a-based biosensing system.
- Designing primers and a reporter system targeting the MP CARDS gene for amplification and fluorescent detection.
- Optimizing reaction conditions for MCDA pre-amplification and CRISPR-Cas12a-gRNA biosensing.
Main Results:
- The MP-MCDA-CRISPR assay demonstrated a limit of detection (LoD) of 50 fg/reaction with no cross-reactivity.
- The assay accurately identified 50 real-time PCR positive and 78 negative clinical samples.
- Optimal MCDA pre-amplification at 64°C and a 5-minute CRISPR-Cas12a-gRNA biosensing time were established.
Conclusions:
- The MP-MCDA-CRISPR assay is a highly promising tool for the rapid diagnosis of MP infection.
- This assay has significant potential for point-of-care (POC) applications in clinical settings.
- The developed platform offers a sensitive and specific method for MP detection.
